1. Field of the Invention
This invention relates to an apparatus and a methodology for measurement of the effect of certain test compounds, such as and without limitation, hormone mimics, on biological signal transduction at the level of biological receptors and their binding to subsequent molecules such as and without limitation DNA molecules, involved in the transduction mechanism. The word "receptor" is defined for purpose of this invention according to the definition appearing in Illustrated Dictionary of Immunology, edited by Julius M. Cruse and Robert E. Lewis and published by CRC Press, Boca Raton, 1995, p.258, ISBN 0-8493-4557-X: "A molecular configuration on a cell or macromolecule that combines with molecules that are complementary to it."
In one embodiment the apparatus and methodology utilizing the principles of the invention are adapted for use as a screening tool for recognizing the presence of estrogen mimics in a sample. In a second embodiment the apparatus and methodology utilizing the principles of the invention are adapted for use as a method for measuring estrogen receptor content in a tissue biopsy sample and evaluating in vitro the probable response of cancer cells, of a type present in that tissue biopsy sample, to certain pharmacologic agents which act through receptor binding. The fiber optic biosensor described herein provides information which reflects the biological impact of the sample. It also has the potential for elucidating mechanisms of receptor action in that the sensor provides a means of obtaining kinetic information on receptor binding events as the binding process occurs.
2. Background of the Invention
Many biological processes are regulated by the binding of regulatory molecules such as hormones, neurotransmitters or cytokines to specific biological receptor molecules. Upon binding to the regulatory molecule, the receptor activates the next step in a signal transduction mechanism by itself binding to another molecular component of the transduction mechanism such as a nuclear response element. The affinity with which this second stage of receptor binding occurs, or in some cases, whether or not this second stage binding occurs at all, is affected by the binding of the regulatory molecule to the receptor. A review of such mechanisms can be found in an article entitled "Mechanisms of Signal Transduction: Sex Hormones, Their Receptors and Clinical Utility" by James L. Wittliff and Wolfgang Raffelsberger, which appeared in Journal of Clinical Ligand Assay, Volume 18, Number 4, Winter, 1995. This text is fully and completely incorporated herein by reference, word for word and paragraph for paragraph.
There are many benefits which derive from the study of both the binding of receptors to regulatory molecules and the second stage binding of the receptors to another component of the signal transduction mechanism. Such study can assist in the design of drugs which exert their biological effect through binding to biological receptors. It can also lead to recognition of compounds in the environment which have the capacity to disrupt important biological regulatory mechanisms by virtue of the ability of such molecules to bind to molecular receptors. It is believed that the presence of such molecules in the environment plays a role in the development of a variety of disease types including cancer, and reproductive problems.
Current methods used for studying these binding phenomena are described in the previously cited review. Because the methods require physical separation of bound from unbound molecules, the methods are quite time consuming and do not have the capacity to provide real time data while binding is occurring between a receptor and a regulatory molecule or between receptors and another component of the signal transduction mechanism. The reliance of current methods on radiolabeled ligands also limits the circumstances under which such measurements can be made.
Evanescent fiber optic sensors provide a method whereby a plurality of molecules of interest bearing an optical tag can be directly monitored as they bind to binding partners attached to an optical fiber. An optical tag may comprise molecules belonging to that class of chemicals which interact with light in a manner so as to alter a characteristic of light received from said sensor by means such as and without limitation, absorbance, fluorescence, luminescence, or polarization; or which produces a second chemical which interacts with light in said manner, such as and without limitation, an enzyme having action producing or destroying a fluorescent, absorbing, luminescent or polarizing compound. The molecular tag may be chemically attached to said plurality of molecules or it may be chemically attached to a plurality of a second type of molecule such as and without limitation, an antibody, having affinity for said molecule of biological interest. Light traveling through an optical fiber at or near the critical angle is totally internally reflected so that it does not significantly effect unbound tagged molecules in the surrounding solution. Total internal reflection does, however, produce an evanescent field which extends about 1000 angstroms from the surface of the fiber. This means that a response from molecules bound to the surface of the fiber can be excited by light totally internally reflected within the fiber, without exciting a response from unbound molecules in the surrounding solution. Therefore measurement of binding can be made without the necessity for physical separation of bound from unbound molecules. Evanescent sensors which measure concentrations of antigen in a solution based upon measurement at a certain time of an amount of fluorescent antigen in the sample which is bound to antibodies on the fiber. Such sensors have been reported in literature and are thoroughly described in the book Biosensors with Fiber Optics, Donald L. Wise and Lemuel B. Wingard, Jr. Editors; Humana Press, Clifton, N.J., 1991. This text is fully and completely incorporated herein by reference, word for word and paragraph for paragraph.
The use of an evanescent sensor having the membrane receptor for acetyl choline bound to its surface to explore the binding of cholinergic ligands has been previously reported by Kim B. Rogers, J. J. Valdes, and Mohyee E. Elderfrawi in an article entitled Acetylcholine receptor fiberoptic evanescent fluorosensor, appearing in the Nov. 1, 1989 issue of Analytical Biochemistry, 182(2):353-9. Membrane receptors for neurotransmitters function differently than receptors for hormones in that membrane receptors do not utilize subsequent binding to a nuclear response element in signal transduction. The transduction process for membrane receptors involves subsequent alteration in transport of ions through channels in the membrane. Use of hormone receptors as reagents in conjunction with an evanescent sensing apparatus possessing a feature resembling a nuclear response element for said hormone receptor has not, to our knowledge, been previously described.
3. Background of the Estrogen Receptor Embodiment
A general background is provided which pertains to the specific embodiment employing the estrogen receptor and the estrogen response element as a means of testing compounds for estrogenic or anti-estrogenic activity. This embodiment is described for the specific application of identification of environmental estrogen mimics and for the specific application of in vitro assessment of the probable efficacy of specific anti-estrogenic compounds, such as and without limitation, tamoxifen, in the treatment of cancer.
The Problems Posed by Estrogen Mimics
During the past 50 years there. has been a marked increase in the number of abnormalities relating to the human reproductive system. Endometriosis, once thought to be a rare condition, has become the most common diagnostic entity in gynecology today. The incidence of female breast cancer is now 1 in 8. A retrospective analysis of data since the 1940's revealed that the sperm count of men all over the world has dropped by 50%, 3 while at the same time testicular cancer rates have tripled and prostate cancer rates have doubled. Furthermore, a 1991 issue of Time magazine reported that one million couples seek treatment for infertility in this country each year. While the above mentioned conditions may seem separate and unrelated, the evidence is that there is one common thread that runs throughout them all: Estrogen Mimics. The evidence warranting concern proved sufficient to cause Congress to pass legislation mandating the EPA to establish a method for evaluating the impact of estrogen mimics in environmental samples.
Current Methods for Measuring the Estrogenic Potency of a Sample
The relationship between the reproductive alterations in humans and the presence of environmental pseudo-estrogens, is difficult to assess because of methodological complexity. One difficulty is that a wide variety of apparently structurally unrelated substances act as estrogen mimics. This makes performing measurements of estrogen mimicry for each possible individual chemical unreasonably time consuming. Additionally, as yet unidentified estrogen mimics which might be present in a sample, could not be measured by chemical or immunologic methods because such methods require knowledge of the identity of the analyte. Although bioassay methods are useful to identify the relative estrogenic activity of pseudo-estrogens, performing bioassays is generally time-inefficient, tedious, and costly.
Because the hER protein is the biological transducer for the first step of estrogen activity, namely binding between the estrogen and the receptor, it possesses the ideal ligand specificity to recognize all estrogenic compounds. This, together with its high affinity (i.e., sensitivity of recognizing small ligand concentrations) make it an ideal probe to combine with biosensor technology.
The Relationship Between Estrogen Receptor and Effective Cancer Treatment
The observation that only 10% of estrogen-receptor negative breast cancer tumors respond to endocrine therapy while 60% of the estrogen-receptor positive tumors respond has made the assessment of estrogen-receptor content of tumors an extremely valuable aid in planning therapeutic course of treatment for breast cancer. Although positive assay for both estrogen-receptor and progesterone receptor increases the likelihood of a positive response to anti-estrogen therapy, still 30-40% of estrogen-receptor positive tumors do not respond to endocrine therapy. A method which will provide improved in vitro assessment of tumor response to endocrine therapy will be of great value in that those patients who require more severe forms of chemotherapy or radiation treatment will receive such treatments initially rather than after endocrine therapy has failed.
Binding between a receptor and its nuclear response element leads to transcription of m-RNA and subsequent protein synthesis. The success of using tamoxifen to treat many ER positive breast cancers illustrates the benefits of targeting molecules involved in these mechanisms of signal transduction and transcriptional regulation.
The Role of Er and Ere in the Biological Response to Estrogens
The initial biological event in estrogen stimulation involves binding of the estrogen or estrogen mimic to the steroid binding region of the receptor.
In a system unchallenged by non-endogenous estrogens, estrogen binds to the estrogen receptor inducing a conformational change that allows for tight binding of the receptor to its recognition element, the ERE, on the DNA. When bound to the DNA this conformation allows for the recruitment of other macromolecules (coactivators, polymerases, kinases, etc.) which, when bound to promoter and TATA regions, incite the transcription of the gene under estrogen dependent regulation. The mRNA is then translated into the proteins responsible for the phenomena which are measured by bioassays.
When non-endogenous estrogens are introduced, the story becomes more varied and much more complex. Many compounds bind to the ER, however, not all are capable of inducing the conformational changes that allow for optimal DNA binding. Those that can bind the receptor to the DNA with a tight affinity introduce an additional wrinkle in that the bound receptor may not be in the proper conformation to recruit the necessary transcription factors. These tightly bound but inactive receptors are occupying many of the ERE binding sites, preventing proper transcription from occurring. This complexity cannot be probed by any single assay that relies on the initiation of transcription for its determination of estrogenisity.
Understanding the Instrument Being Utilized
The preferred embodiments of this invention utilize the estrogen receptor (ER) and the estrogen response element (ERE) as the recognition elements in an evanescent fiber-optic sensor to produce an instrument for measuring the potential of chemicals, environmental contaminants, and pharmaceuticals to interfere with estrogen mediated biological processes. Understanding of the instrument requires an understanding of (1) the principle of operation of evanescent fiber optic sensors and (2) the role of these two molecules in estrogen mediated biological responses.
The Principle of Operation of Evanescent Sensors
The essential feature of an evanescent biosensor, is confinement of the measurement area to the surface of the waveguide by taking advantage of the evanescent field associated with total internal reflection within the fiber. This was originally described in the context of immunoassay by Tomas Hirshfield in U.S. Pat. No. 4,447,546 entitled "Fluorescent immunoassay employing optical fiber in a capillary tube" which is herein incorporated by reference, line by line and word for word. The manner in which this functions is as follows.
Consider light incident at angle q on the boundary between two optical media with indexes of refraction N and n (N&gt;n). When the light is incident on the boundary at angles greater than or equal to the critical angle, .theta..sub.crit where sin(.theta..sub.crit)=n/N, the light will be totally reflected from the surface. Although, light is not transmitted past the boundary and into the media with the lower index of refraction, electromagnetic theory shows that an evanescent electromagnetic field decays exponentially with perpendicular distance from the boundary. The characteristic 1/e depth of this decay for light of wavelength .lambda. incident at angle .theta. is given by the equation: EQU (.lambda.4.pi.)(N.sup.2 sin.sup.2.theta.-n.sup.2).sup.-1/2. Equation 1
This distance is large compared with the dimensions of proteins and biologically significant nucleotides. Thus, the light with wavelength .lambda..sub.1 will interact with fluorescent molecules, which are associated with any proteins or nucleotides that are attached near the probe's surface, to generate fluorescence at wavelength .lambda..sub.2. Because the waveguide is very large compared with the size of the proteins or nucleotides, a large fraction of the emitted fluorescence light at wavelength .lambda..sub.2 will intersect the fiber optic sensor, then be trapped inside due to total internal reflection, and finally be carried back to a solid state light detector in the control unit.